JP7240679B2 - multiple sclerosis marker - Google Patents

Description

The present invention relates to a method for testing a demyelinating disease (especially multiple sclerosis), and more specifically, a method for detecting a protein tyrosine phosphatase receptor-type Z (PTPRZ) in a sample derived from a subject, More preferably, it relates to a method for testing multiple sclerosis, which comprises the step of measuring branched O-mannose sugar chain-modified PTPRZ.

Multiple sclerosis (MS) is one of demyelinating diseases in which multiple demyelinating lesions appear in the white matter of the central nervous system and cause neurological disorders such as motor paralysis and sensory disturbance. It is common in Caucasians in Europe and the United States, and in Northern Europe, it occurs in 50 to 100 out of 100,000 people. In Japan, there are only about 10 out of 100,000 people (Non-Patent Document 1), but the number of patients is increasing year by year, and the number of patients worldwide has reached 2.5 million or more.

The clinical picture of multiple sclerosis is roughly divided into (1) relapsing-remitting MS (RRMS), (2) primary progressive MS (PPMS), (3) secondary progressive ( secondary progressive MS; SPMS). RRMS is characterized by alternating episodes of worsening symptoms (relapses) and stable symptoms (remissions), with periods of remission and relapses of several months to several years. PPMS is characterized by a temporary stagnation period during which the disease does not progress, but the disease progresses gradually without remission. SPMS is characterized by repeated relapses and remissions in the early stage of onset, followed by gradual progress. At present, no curative therapy has been established for multiple sclerosis either, and symptomatic therapy is performed according to the time of onset and symptoms of the patient.

One of the factors that make the treatment of multiple sclerosis difficult is the difficulty of its diagnosis. Multiple sclerosis is difficult to diagnose by doctors because it has various symptoms and no biomarkers specific to the disease have been found. In particular, when the lesion is solitary, it is difficult to distinguish it from a brain tumor, encephalitis, or encephalopathy. Currently, multiple sclerosis is diagnosed by MRI examination and cerebrospinal fluid examination. A definitive diagnosis of multiple sclerosis can be made only after excluding the possibility of other diseases by attempting tissue biopsy of the affected area, which may be dangerous in some cases (Non-Patent Document 7). Multiple sclerosis is treated with steroids to suppress the immune system, while brain tumors are treated with a combination of surgery, radiotherapy, and drug therapy. Accurate diagnosis is important because treatment approaches differ for each disease. Despite this, early diagnosis of multiple sclerosis is currently very difficult and, in fact, the time between the appearance of the first symptoms of multiple sclerosis and the definitive diagnosis of multiple sclerosis is very high. Some survey results indicate that the average period exceeds three years.

Against this background, there is a strong demand for the establishment of a test method that can more easily diagnose multiple sclerosis.

Receptor-type protein tyrosine phosphatases are classified into eight subfamilies based on structural similarity (Non-Patent Document 2). Its extracellular region is composed of various domains such as immunoglobulin-like (Ig) domain, fibronectin-like (FN) domain, carbonic anhydrase-like (CAH) domain, and Meprin-A5-PTPμ (MAM) domain. , there are one or two tyrosine phosphatase (PTP) domains in the cell. In the tandem receptor-type protein tyrosine phosphatase, only D1 on the proximal side of the cell membrane has phosphatase activity, and D2 has no enzymatic activity (exceptionally, some activity is observed in D2 of the R4 RPTP subfamily). ).

Receptor-type protein tyrosine phosphatase Z (Protein Tyrosine Phosphatase Receptor-type Z1; also called PTPRZ, PTPRZ1, or Ptprz, PTPζ or RPTPβ, etc.) belongs to the R5 subfamily. Mammalian PTPRZ has splicing variants and, for example, in mice this gives rise to three isoforms (PTPRZ-A and PTPRZ-B, which are receptor forms, and PTPRZ-S, which is a secreted form). PTPRZ is composed of an extracellular CAH domain, an FN type III-like domain, a chondroitin sulfate chain-binding domain (rich in Ser-Gly/Gly-Ser) and two intracellular PTP domains (D1 and D2). . Unlike other RPTPs, PTPRZ has sulfated polysaccharide modifications called sulfated glycosaminoglycans such as chondroitin sulfate and keratan sulfate in the extracellular domain. In brain tissue, which is the major expression site of PTPRZ, all isoforms are expressed as chondroitin sulfate proteoglycans (Non-Patent Documents 3 to 5). PTPRZ is expressed on both neuronal and glial cells (astrocytes and oligodendrocytes) in the central nervous system. PTPRZ is also called N-acetylglucosaminyltransferase GnT-IX (MGAT5B, Mannosyl (Alpha-1,6-)-Glycoprotein Beta-1,6-N-Acetyl-Glucosaminyltransferase, Isozyme B) in response to demyelinating stimuli. It has been reported that it undergoes branched O-mannose modification via (non-patent document 6).

Kinoshita M. et al., Neurol Sci. 2015;36(7):1147-51. Tonks NK, Nat Rev Mol Cell Biol. 2006 Nov;7(11):833-46. Nishiwaki T. et al., J Biochem. 1998 Mar;123(3):458-67. Chow JP. et al., J Biol Chem. 2008 Nov 7;283(45):30879-89. Chow JP. et al., Neurosci Lett. 2008 Sep 19;442(3):208-12. Kanekiyo K. et al., J Neurosci. 2013 Jun 12;33(24):10037-47. Nakazawa Y. et al., Jpn. J. Cli. Immunol.2013;36(3):175-9

An object of the present invention is to provide a novel examination method for demyelinating diseases (especially multiple sclerosis).

As a result of intensive studies on the above problems, the present inventors found secretory (free) PTPRZ isoforms corresponding to the extracellular regions of PTPRZ isoforms 1 and 2 in the cerebrospinal fluid of demyelinating disease patients. It was found that the content of Forms 1 and 2 was reduced compared to that of the control group. In particular, we found that the content of secretory PTPRZ isoforms 1 and 2 in the cerebrospinal fluid of patients with multiple sclerosis was significantly reduced. Furthermore, surprisingly, it was found that the amount of branched O-mannose glycosylation-type PTPRZ produced by glycosylation of PTPRZ by glycosyltransferase GnT-IX was also reduced in the cerebrospinal fluid compared to the control group. Based on these findings, the present invention has been completed by further research. That is, the present invention is as follows.

[1] A method for testing multiple sclerosis, comprising the step of measuring secretory receptor protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject, wherein the secretory PTPRZ in the sample derived from the subject If the measured amount is low compared to the amount of secreted PTPRZ in a control group sample, the subject is indicated to have multiple sclerosis, and the secreted PTPRZ is a secreted PTPRZ isoform. 1 and secreted PTPRZ isoform 2 or both.
[2] The method according to [1], wherein the secretory PTPRZ isoform 1 and the secretory PTPRZ isoform 2 are branched O-mannose sugar chain-modified PTPRZ.
[3] The method of [1] or [2], wherein the subject-derived sample is cerebrospinal fluid or blood.
[4] Measurement of secretory PTPRZ in spinal fluid derived from a subject is performed using an antibody that specifically recognizes the protein or an antibody that specifically recognizes a branched O-mannose sugar chain. The method according to any one of [1] to [3].
[5] A diagnostic kit for multiple sclerosis comprising either or both of an antibody that specifically recognizes a secretory PTPRZ and an antibody that specifically recognizes a branched O-mannose sugar chain, wherein the secretory A kit, wherein the PTPRZ is either or both of secreted PTPRZ isoform 1 and secreted PTPRZ isoform 2.
[6] A method for examining a state of demyelination, comprising the step of measuring secretory receptor protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject, wherein the amount of secretory PTPRZ in the sample derived from the subject is low compared to the amount of secreted PTPRZ in the control group sample, indicating that the subject is demyelinating, and the secreted PTPRZ is secreted by secreted PTPRZ isoform 1 and secreted PTPRZ isoform 2, or both.
[7] A method for testing a demyelinating state, comprising the step of measuring branched O-mannose sugar chain-modified receptor protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject, wherein When the measured amount of branched O-mannose glycosylation-type PTPRZ in the sample is lower than the amount of branched O-mannose glycosylation-type PTPRZ in the sample of the control group, the subject is demyelinated. and the branched O-mannose sugar chain-modified PTPRZ is the branched O-mannose sugar chain-modified PTPRZ isoform 1 and the branched O-mannose sugar chain-modified PTPRZ isoform 2. A method that is either or both.

In another aspect, the present invention is as follows.
[1A] A method for testing multiple sclerosis, comprising the step of measuring receptor-type protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject, wherein the amount of secretory PTPRZ in the sample derived from the subject A method wherein a measured value that is low compared to the amount of PTPRZ in a control group sample indicates that the subject has multiple sclerosis.
[2A] The method of [1A], wherein the PTPRZ is a branched O-mannose sugar chain-modified PTPRZ.
[3A] The method of [1A] or [2A], wherein the subject-derived sample is cerebrospinal fluid or blood.
[4A] PTPRZ in spinal fluid derived from a subject is measured using an antibody that specifically recognizes the protein or an antibody that specifically recognizes a branched O-mannose sugar chain. , the method according to any one of [1A] to [3A].
[5A] PTPRZ is one or more selected from the group consisting of secretory PTPRZ isoform 1, secretory PTPRZ isoform 2, and secretory PTPRZ isoform 3, [1A] to [ 4A].
[6A] A diagnostic kit for multiple sclerosis containing either or both of an antibody that specifically recognizes PTPRZ and an antibody that specifically recognizes a branched O-mannose sugar chain.
[7A] A method for testing a demyelinating state, comprising the step of measuring receptor-type protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject, wherein the amount of secretory PTPRZ in the sample derived from the subject is measured. A method wherein a low value compared to the amount of PTPRZ in a control group sample indicates that the subject is demyelinating.
[8A] A method for testing a demyelinating state, comprising the step of measuring branched O-mannose sugar chain-modified receptor protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject, wherein When the measured amount of branched O-mannose glycosylation-type PTPRZ in the sample is lower than the amount of branched O-mannose glycosylation-type PTPRZ in the sample of the control group, the subject is demyelinated. A method that is shown to be causing

The present invention provides a new objective index useful for definitive diagnosis of multiple sclerosis.

FIG. 2 is a conceptual diagram showing the structures of three isoforms of human PTPRZ. A indicates isoform 1, B indicates isoform 2, and C indicates isoform 3. In the figure, CA represents a carbonic anhydrase-like domain, FN a type III fibronectin-like domain, CS a chondroitin sulfate chain-binding region, TM a transmembrane domain, and D1 and D2 both PTP domains. FIG. 2 shows the results of immunohistochemical staining using Cat-315 antibody or anti-PTPRZ antibody in multiple sclerosis patient brain sections. Figure 3 shows that in patients with multiple sclerosis, the amount of secretory (free) PTPRZ isoform 1 and secretory (free) PTPRZ isoform 2 in the cerebrospinal fluid of patients with idiopathic normal pressure hydrocephalus (control group) ) is significantly reduced compared to that of . In addition, data were shown by the average value + standard error (SE).

The present invention will be described in detail below.

1. Method for testing multiple sclerosis The present invention comprises the step of measuring either or both of secretory receptor protein tyrosine phosphatase Z (PTPRZ) isoforms 1 and 2 in a sample derived from a subject. (hereinafter sometimes referred to as the "inspection method of the present invention").

Multiple sclerosis, which can be tested for presence or absence by the test method of the present invention, can be classified into three types as described above. According to the present invention, any type of relapsing-remitting disease (RRMS), primary progressive disease (PPMS), and (3) secondary progressive disease (SPMS) can be tested.

In one aspect of the testing method of the present invention, the content of either or both of the secretory PTPRZ isoforms 1 and 2 in a sample derived from a subject is measured as an index, and compared with the corresponding values in the control group. , the presence or absence of multiple sclerosis in a subject can be examined. In humans, PTPRZ has three types of splicing variants that differ in the length of the extracellular region. Number 3). Furthermore, a secreted (or "free") PTPRZ is known, which is produced by protease cleavage of the extracellular region of each isoform. A splicing variant lacking the C-terminal region after the transmembrane region has also been reported in rodents, but the corresponding variant in humans has not been identified. Therefore, in the test method of the present invention, the content of secretory (free) PTPRZ isoform 1 and/or 2 in a sample derived from a subject is measured, and a control group (healthy subjects or those suffering from multiple sclerosis The presence or absence of multiple sclerosis in the subject can be examined by comparing with the corresponding values of subjects who have not been tested. Each isoform of human PTPRZ and their secretory forms are described in detail below. As used herein, the term "secreted (free) PTPRZ" refers to a polypeptide fragment derived from the extracellular region of each isoform of human PTPRZ. PTPRZ has a highly protease-sensitive site on the C-terminal side of the extracellular domain. This site is cleaved by a protease such as a metalloprotease, resulting in secreted (free) PTPRZ. As used herein, the "secreted" PTPR isoform can be interchanged with the "free" PTPRZ isoform.

PTPRZ isoform 1 consists of 2315 amino acids shown in SEQ ID NO:1. PTPRZ isoform 1 has a signal peptide at positions 1-24, a CA domain at positions 45-298, an FN domain at positions 313-401, a transmembrane domain at positions 1637-1662, and a D1 domain at positions 1717-1992. and a D2 domain at positions 2023-2282. In addition, the CS binding region corresponds to the region where the CS chain binds between the FN domain and the transmembrane domain. The extracellular region of PTPRZ isoform 1 consists of the amino acid sequence shown in SEQ ID NO:4 corresponding to positions 25-1636 in the amino acid sequence shown in SEQ ID NO:1. Full-length or partial fragment polypeptides of SEQ ID NO:4 correspond to secreted PTPRZ isoform 1.

PTPRZ isoform 2 consists of 2308 amino acid residues shown in SEQ ID NO:2. PTPRZ isoform 2 is an isoform in which 7 amino acid residues corresponding to positions 1723-1729 of the D1 domain in PTPRZ isoform 1 are deleted. Therefore, the extracellular region of isoform 2 matches that of isoform 1 and consists of the amino acid sequence shown in SEQ ID NO:4. Therefore, the full-length or partial fragment polypeptide of SEQ ID NO: 4 corresponds to secretory PTPRZ isoform 2.

PTPRZ isoform 3 consists of 1448 amino acid residues shown in SEQ ID NO:3. PTPRZ isoform 3 is an isoform lacking 860 amino acid residues at positions 755-1614, which may correspond to most of the CS binding region in PTPRZ isoform 1, and 7 amino acid residues corresponding to positions 1723-1729. be. Therefore, the extracellular region of isoform 3 is shorter than that of isoforms 1 and 2 and consists of amino acid sequences corresponding to positions 25-754 and 1615-1636 in the amino acid sequence shown in SEQ ID NO:1.

There are two types of secretory PTPRZ: a long-form of about 350 kDa derived from the extracellular domains of PTPRZ isoform 1 and PTPRZ isoform 2, and a short-form of about 180 kDa derived from the extracellular domain of PTPRZ isoform 3. Among them, the long-form (and its fragment polypeptide) can be a marker for detection of multiple sclerosis. In the present invention, the amino acid sequence to be actually detected is a full-length polypeptide of the amino acid sequence (1612 amino acids) shown in SEQ ID NO: 4 and/or a partial peptide consisting of a portion thereof. Such partial peptides include, for example, peptides containing 1129 or more consecutive amino acids, peptides containing 1209 or more consecutive amino acids, and 1290 or more consecutive amino acids of the amino acids shown in SEQ ID NO:4. peptides, peptides containing a continuous amino acid sequence of 1371 or more amino acids, peptides containing a continuous amino acid sequence of 1451 or more amino acids, peptides containing a continuous amino acid sequence of 1532 or more amino acids, peptides containing a continuous amino acid sequence of 1580 or more amino acids, Also, peptides containing a continuous amino acid sequence of 1596 or more amino acids are exemplified, but not limited to these.

Further, as another aspect of the method of the present invention, by measuring the content of branched O-mannose in a sample derived from a subject and comparing it with the corresponding value in the control group, multiple occurrence in the subject The presence or absence of sclerosis can be tested. PTPRZ undergoes a branched O-mannose modification via N-acetylglucosaminyltransferase GnT-IX in response to demyelinating stimuli. Therefore, the content of branched O-mannose in a subject-derived sample is thought to reflect the abundance of secreted PTPRZ isoforms 1 and 2. Therefore, the presence or absence of multiple sclerosis can also be detected by measuring the content of branched O-mannose in a sample derived from a subject and comparing it with the corresponding value in a control group.

As a method for measuring secretory PTPRZ isoforms 1 and 2 in a sample derived from a subject, any method may be used as long as the amount of the protein can be quantified. Examples include, but are not limited to, mass spectrometry, chromatography (liquid chromatography, gas chromatography, etc.), electrophoresis, immunoassay using antibodies, and the like. In particular, the immunoassay method is simple and preferable. Immunoassays are classified into sandwich method, competition method, agglutination method, western blotting method, etc. according to the reaction format, and radioimmunoassay, fluorescence immunoassay, enzyme immunoassay (EIA) according to labeling. , biotin immunoassays, and the like, but are not limited to these.

Antibodies used for immunoassay may be either polyclonal antibodies or monoclonal antibodies, but monoclonal antibodies are preferred from the viewpoint of reproducibility. In carrying out the testing method of the present invention, a commercially available antibody may be used, and secretory PTPRZ isoforms 1 and/or 2, or branched O-mannose sugar chain-modified PTPRZ isoforms may be tested by a method known per se. Antibodies that specifically recognize 1 and/or 2 may be produced and used.

The term "antibody" as used herein is a concept that also includes "antigen-binding fragment". “Antigen-binding fragment” means an antibody fragment that maintains the binding (antigen-antibody reactivity) to the corresponding antigen of the original antibody. Examples of such antigen-binding fragments include, but are not limited to, Fab, F(ab') ₂ , scFv, and the like. Such antigen-binding fragments can be prepared by a method known per se. Examples of commercially available antibodies used in the testing method of the present invention include, but are not limited to, mouse monoclonal Cat-315 IgM (Millipore) and mouse anti-PTPRZ IgMκ (122.2) (Santa Cruz biotechnology).

As used herein, the expression that an antibody "specifically recognizes" an antigen means that _{the KD} value for the binding affinity of the antibody to the antigen in the antigen-antibody reaction is 1×10 ⁻⁵ M or less (preferably 1× 10 ⁻⁶ M or less, 1×10 ⁻⁷ M or less, 1×10 ⁻⁸ M or less, more preferably 1×10 ⁻⁹ M or less, most preferably 1×10 ⁻¹⁰ M or less). .

Examples of antibodies used for measuring secretory PTPRZ isoforms 1 and/or 2 include the following antibodies:
(1) Antibody that specifically recognizes secretory PTPRZ isoform 1 (2) Antibody that specifically recognizes secretory PTPRZ isoform 2 (3) Antibody that specifically recognizes secretory PTPRZ isoforms 1 and 2 .
In addition, PTPRZ is modified with a branched O-mannose sugar chain in vivo by GnT-IX, which is a glycosyltransferase. As detailed in the examples below, we demonstrate that secretory PTPRZ isoforms 1 and 2 in cerebrospinal fluid are also branched O-mannose glycosylated. . Therefore, instead of the anti-PTPRZ antibody, an antibody such as the Cat-315 antibody that specifically recognizes the branched O-mannose sugar chain on PTPRZ can also be used in the test method of the present invention. These antibodies can be prepared by methods known per se, and commercially available ones may also be used. From such a point of view, the testing method of the present invention can be defined as follows: branched O-mannose glycosylation-type receptor protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject A method for testing multiple sclerosis, comprising the step of measuring, wherein the measured value of the amount of branched O-mannose glycosylation-type PTPRZ in the sample derived from the subject is equal to the amount of branched O-mannose sugar chain-modified PTPRZ in the sample of the control group - a method, wherein a low relative to mannose glycosylated PTPRZ amount indicates that the subject has multiple sclerosis.

In the test method of the present invention, the amount of secretory PTPRZ isoforms 1 and/or 2 in a sample derived from a subject is measured. Although cerebrospinal fluid is used as a sample in the following examples, blood can also be used because cerebrospinal fluid is known to flow into veins. In preferred embodiments, the subject-derived sample is cerebrospinal fluid.

In the test method of the present invention, when the measured value of secretory PTPRZ isoform 1 and / or 2 in the sample derived from the subject is significantly lower than the amount of the protein in the sample of the control group Shown to be sclerosis. In the comparison of the measured values, a cutoff value is set in advance, and the cutoff value is compared with the measured value of the amount of secretory PTPRZ isoforms 1 and/or 2 in the sample derived from the subject. good too. A measured value below the cutoff value indicates that the subject is suffering from multiple sclerosis.

A person skilled in the art can set the cutoff value using a method known per se. For example, multiple samples are obtained from known patient and control groups (healthy subjects or subjects not suffering from multiple sclerosis) and the amounts of secreted PTPRZ isoforms 1 and/or 2 are measured and desirably A cut-off value may be set at which the sensitivity and/or specificity can discriminate between the two groups.

Examples of subjects to which the test method of the present invention is applied include mammals such as humans, monkeys, rats, dogs, and cats, with humans being particularly preferred.

2. A test kit for multiple sclerosis The present invention also provides an antibody that specifically recognizes either or both of the secretory PTPRZ isoforms 1 and 2 and an antibody that specifically recognizes a branched O-mannose sugar chain. A diagnostic kit for multiple sclerosis (hereinafter sometimes referred to as "the kit of the present invention") containing either or both of them is provided. Using the kit of the present invention, the amount of secretory PTPRZ isoforms 1 and/or 2 in a sample derived from a subject is measured and compared with the corresponding amount in a control group sample to determine whether the subject is overrepresented. It is possible to easily test whether or not a person is suffering from sexual sclerosis.

Antibodies specifically recognizing secretory PTPRZ isoforms 1 and/or 2 contained in the kit of the present invention are exemplified by the following antibodies:
(1) Antibody that specifically recognizes secretory PTPRZ isoform 1 (2) Antibody that specifically recognizes secretory PTPRZ isoform 2 (3) Antibody that specifically recognizes secretory PTPRZ isoforms 1 and 2 (4) Antibodies that specifically recognize branched O-mannose sugar chains.
(4) An antibody that specifically recognizes a branched O-mannose sugar chain can be prepared using a method known per se, and as described above, using a commercially available antibody such as the Cat-315 antibody. good too. In one aspect, the kit of the present invention comprises one or more of the above antibodies (1) to (4) as components. In addition, the kit of the present invention may contain components other than the antibody, such as instruction manuals and evaluation criteria.

In one aspect of the present invention, the multiple sclerosis testing method of the present invention may be combined with a multiple sclerosis treatment method. Accordingly, the present invention provides methods for testing and treating multiple sclerosis, comprising the steps of:
(1) a step of measuring secretory receptor protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject, wherein the measured value of the amount of secretory PTPRZ in the sample derived from the subject is equal to a control A low compared to the amount of secreted PTPRZ in the group samples indicates that the subject has multiple sclerosis, and the secreted PTPRZ comprises secreted PTPRZ isoform 1 and secreted PTPRZ isoform and (2) administering a therapeutically effective amount of a therapeutic agent for multiple sclerosis to the subject determined to have multiple sclerosis in step (1). .

Examples of therapeutic agents for multiple sclerosis used in the present invention include steroids, interferon β, S1P receptor modulators, immunosuppressants, alkylating agents, anticancer antibiotics, anti-CD52 antibodies, and anti-α4 integrin. Administration of therapeutic agents for multiple sclerosis, such as antibodies, includes, but is not limited to.

Steroids include, for example, amcinonide, hydrocortisone sodium succinate, prednisolone sodium succinate, prednisolone methylsuccinate, ciclesonide, difluprednate, betamethasone propionate, dexamethasone, deflazacort, triamcinolone, triamcinolone acetonide, halcinonide, dexamethasone palmitate, hydrocortisone , flumethasone pivalate, prednisolone butylacetate, budesonide, prasterone sulfate, mometasone furoate, fluocinonide, fluocinolone acetonide, fludroxycortide, flunisolide, prednisolone, alclomethasone propionate, clobetasol propionate, dexamethasone propionate, propionic acid Deprodone, fluticasone propionate, beclomethasone propionate, betamethasone, methylprednisolone, methylprednisolone leptanate, methylprednisolone sodium succinate, dexamethasone sodium phosphate, hydrocortisone sodium phosphate, prednisolone sodium phosphate, diflucortolone valerate, gilt dexamethasone herbate, betamethasone valerate, prednisolone acetate valerate, cortisone acetate, diflorasone acetate, dexamethasone acetate, triamcinolone acetate, paramethasone acetate, halopredone acetate, fludrocortisone acetate, prednisolone acetate, methylprednisolone acetate, clobetasone butyrate, hydrocortisone butyrate, propionate butyrate Acid hydrocortisone, betamethasone butyrate propionate.

S1P receptor modulators include, for example, fingolimod, siponimod, ponesimod, ceralifimod, ozanimod.

Immunosuppressants include, for example, azathioprine, ascomycin, everolimus, salazosulfapyridine, cyclosporine, cyclophosphamide, sirolimus, tacrolimus, bucillamine, methotrexate, leflunomide, teriflunomide.

Alkylating agents include, for example, nitrogen mustard-N-oxide hydrochloride, cyclophosphamide, ifosfamide, melphalan, thiotepa, carbocone, busulfan, nimustine hydrochloride, dacarbazine, lanimustine, carmustine, chlorambucil, bendamustine, mecloethanamine. .

Anticancer antibiotics include, for example, actinomycin D, mitomycin C, daunorubicin hydrochloride, doxorubicin hydrochloride, aclarubicin hydrochloride, neocarzinostatin, pirarubicin hydrochloride, epirubicin (hydrochloride), idarubicin hydrochloride, chromomycin A3, bleomycin (hydrochloride) , peplomycin sulfate, terarubicin, dinostatin stimaramer, gemtuzumab ozogamicin, mitoxantrone hydrochloride.

Anti-CD52 antibodies include, for example, alemtuzumab. Anti-α4 integrin antibodies also include, for example, natalizumab.

The therapeutically effective amount of the therapeutic agent for multiple sclerosis may vary depending on the type of active ingredient, the body weight and sex of the subject to be treated, the route of administration, etc., but an appropriate dosage and administration method can be appropriately selected by those skilled in the art. can do. The route of administration of the therapeutic agent can be either oral or parenteral, depending on the type of active ingredient. Parenteral administration includes, but is not limited to, subcutaneous administration, intramuscular administration, intraperitoneal administration, and the like.

"Treatment" of a disease as used herein may include not only cure of the disease, but also remission of the disease and amelioration of the severity of the disease.

The present invention will be described in more detail in the following examples, but the present invention is not limited by these examples.

All animal experiments were conducted in accordance with the RIKEN Animal Experiment Code. Immunohistological analysis of human cerebrospinal fluid and brain was performed at Fukushima Medical University according to the university's protocol. Usually, when lumbar puncture is performed to collect cerebrospinal fluid, headache may occur after the puncture, and if the symptoms are severe, it may be accompanied by slight fever, headache, and vomiting. Collect cerebrospinal fluid only for the patient.

[Example 1] Immunohistochemical staining A paraffin-embedded section of the brain of a patient with multiple sclerosis was deparaffinized as follows. It was immersed in xylene three times, 99.5% ethanol three times, 90% ethanol once, 80% ethanol once, and 70% ethanol once for 5 minutes each, and then immersed in running water for 5 minutes. It was autoclaved in 0.01 M citrate buffer (pH 8.5) at 121° C. for 5 minutes and immersed in PBS (pH 7.8).

After washing sections of multiple sclerosis patient brains with PBS, they were permeabilized by soaking in 0.5% Triton X-100-PBS for 15 minutes. After washing in 0.05% Tween-20-PBS (PBS-T) with shaking three times for 5 minutes each, the cells were shaken in 5% normal goat serum PBS for 30 minutes. After washing twice for 5 minutes each in PBS-T, primary antibodies (mouse monoclonal Cat-315 IgM (Millipore), mouse anti-PTPRZ IgMκ (122.2) (Santa cruz biotechnology) diluted with Dako REAL antibody diluent (Dako) for 2 hours at room temperature or overnight at 4° C. After washing three times for 5 minutes each in dilution ratio PBS-T, Alexa fluor 546- or 488-labeled secondary antibody and DAPI diluted with DAKO REAL antibody diluent. The cells were immersed in room temperature for 45 minutes, washed three times in PBS-T for 5 minutes each, mounted in CC/Mount, and observed with an FV1000-D confocal laser microscope (Olympus). Intensity analysis was performed by MetaMorph (Olympus) (Fig. 1), which revealed that Cat-315-positive cells and PTPRZ-positive cells were consistent, and that Cat-315-positive cells were PTPRZ-positive. rice field.

[Example 2]
Cerebrospinal fluid from patients with multiple sclerosis, cerebrospinal fluid from patients with neuromyelitis optic (NMO), and idiopathic normal pressure hydrocephalus (iNPH) were suspected. Subjects diagnosed as not having iNPH (non-iNPH, used as a control group) were used as multiple sclerosis disease controls. These cerebrospinal fluids were patient samples obtained at medical institutions, mainly Fukushima Medical University Hospital, and informed consent was obtained from all patients. Antibodies used were mouse monoclonal Cat-315 IgM (Millipore), mouse anti-PTPRZ IgMκ (122.2) (Santa Cruz biotechnology), goat polyclonal anti-Transthyretin (TTR) antibody (Abcam). Other reagents were mainly purchased from Wako.

10 μL of cerebrospinal fluid derived from MS patients (n=3), NMO patients (n=3) and non-iNPH patients (n=3, control) was subjected to chondroitinase digestion and Western blotting. The details of chondroitinase digestion are as follows. A final concentration of 50 mM Tris-HCl (pH 8.0), 30 mM sodium acetate, and 200 U/L Chondroitinase ABC (Seikagaku) were added to the sample solution and stirred at 37° C. for 1 hour.

Western blotting was performed according to the following procedure. Laemmli's sample buffer (final concentration 20.8 mM Tris-HCl (pH 6.5), 8.3% glycerol, 1% SDS, 1% β-mercaptoethanol) was added to the sample solution and heated at 99° C. for 5 minutes. A 3-10% gradient polyacrylamide gel (Atto) was run at a constant current of 20-25 mA per gel for 60-70 minutes. Using a semi-dry transfer device, transfer was performed on the nitrocellulose membrane at a constant current of 150 mA for 90 minutes. After blocking by soaking in 5% skim milk-0.05% Tween 20-TBS (pH 7.4) (TBS-T) for 30 minutes, the cells were placed in primary antibody diluted with TBS-T for 2 hours at room temperature or 4°C. and shaken overnight. After being immersed and washed three times in TBS-T for 5 minutes each, they were reacted with an HRP-labeled secondary antibody diluted in TBS-T for 40 minutes at room temperature. After washing three times with TBS-T, light was emitted using a SuperSignal West Femto (Thermo Fisher Scientific) and the signal was detected with a LAS4000 mini (GE Healthcare). The quantification of the band signal intensity was performed using the quantification software attached to the LAS4000 mini. Table 1 shows the results. Table 1 shows the signal intensity of the upper band of secretory PTPRZ isoforms 1 and 2 relative to the signal intensity of TTR detected as an internal standard of cerebrospinal fluid in MS patient-derived, MNO patient-derived, and non-iNPH-derived samples. It is shown as a relative value of the signal intensity of the upper band detected with the Cat-315 antibody.

As shown in Table 1, in multiple sclerosis patient-derived cerebrospinal fluid, the upper band (PTPRZ isoform 2 full length is PTPRZ isoform 1 full length and Since there is only a difference of 7 amino acids and the amino acid sequences of the extracellular domains of both are identical, they are detected as the same band (approximately 350 kDa) and the lower row, which is thought to be derived from the extracellular domains of the secretory PTPZ isoform 3. A band (approximately 180 kDa) signal was detected. Among these, it was found that the cerebrospinal fluid derived from patients with multiple sclerosis showed a marked decrease in the amount of signal in the upper band compared to the control. In addition, the signal amount of the corresponding branched O-mannose sugar chain-modified PTPRZ was similarly decreased. Interestingly, some of the samples from NMO patients had decreased levels to the same levels as those from MS patients. NMO is a disease in which inflammation occurs when astrocyte aquaporin 4 is attacked by autoantibodies, and it is believed that demyelination occurs as inflammation progresses. Therefore, the fact that branched O-mannose glycosylation-type PTPRZ was decreased in some NMO patient specimens may indicate that demyelination occurs in these patients as in MS. Conceivable. From the above, it was shown that the amount of branched O-mannose sugar chain-modified PTPRZ in cerebrospinal fluid can be used as a biomarker reflecting the degree of demyelination.

[Example 3] Verification of secretory PTPRZ isoforms 1 and 2 in cerebrospinal fluid as MS detection markers Through the following experiments, secretory PTPRZ (secretory PTPRZ isoforms 1 and 2 (that is, secretory PTPRZ long- form)) can function as a marker for MS detection.

Secretory PTPRZ isoform 1 in cerebrospinal fluid derived from multiple sclerosis (MS) patient group (24 patients) and idiopathic normal pressure hydrocephalus (iNPH) patient (including suspected cases) group (25 patients) and 2 were determined by Western blotting.

Anti-PTPRZ antibody mouse anti-PTPζ IgMκ (sc-33664, Santa Cruz) and goat anti-mouse IgM antibody-HRP (SAB-110, Stressgen) were used as antibodies. Mouse anti-PTPZ IgMκ is a PTPRZ-specific monoclonal antibody produced by preparing proteoglycan fractions from fetal rat brain. Other reagents were mainly purchased from Wako.

Ten microliters of cerebrospinal fluid collected from each of the above patients was first subjected to chondroitinase digestion. Specifically, 100 mM Tris-HCl (pH 7.5), 60 mM sodium acetate, and 4 mU chondroitinase ABC (Sigma, 2905) were added to the sample solution and incubated at 37° C. for 1 hour.

After that, Western blotting was performed. Western blotting was performed according to the following procedure. Laemmli's sample buffer (final concentration 20.8 mM Tris-HCl (pH 6.5), 8.3% glycerol, 1% SDS, 1% β-mercaptoethanol) was added to the chondroitinase-treated sample solution and heated for a minute. A 3-10% gradient polyacrylamide gel (Atto, NPG-310L) was run at a constant current of 20-25 mA per gel for 60-70 minutes. Using a wet transfer device, transfer was performed on the nitrocellulose membrane at a constant current of 350 mA for 45 minutes. After blocking by immersing in 1% BSA-PBS (pH 7.4) overnight at 4°C, mouse anti-PTPRZ IgMκ (Santa cruz, sc-33664) as a primary antibody and shaken at room temperature for 2 hours in 1.5 mL of PBS-T. Subsequently, after being immersed in PBS-T three times for 5 minutes each and washed, PBS-T containing 10,000-fold diluted HRP-labeled goat anti-mouse IgM antibody (SAB-110, Stressgen) as a secondary antibody. and reacted at room temperature for 2 hours. After washing three times with TBS-T, color was developed using SuperSignal West Femto maximum sensitivity substrate (Thermo Fisher Scientific, 34096), and the resulting signal was detected with ATTO Ez-Capture MG (ATTO). The quantification of the band signal intensity was performed using the attached quantification software CS Analyzer. The results are shown in FIG.

As shown in FIG. 3, the signal intensities of secretory PTPRZ isoforms 1 and 2 in the cerebrospinal fluid derived from MS patients were higher than those of the cerebrospinal fluid from the control group (patients with idiopathic normal pressure hydrocephalus (including suspected cases)). significantly lower than that in the These results indicated that secretory PTPRZ isoforms 1 and 2 can serve as MS detection markers.

INDUSTRIAL APPLICABILITY According to the present invention, a new objective index useful for definitive diagnosis of demyelinating diseases (particularly multiple sclerosis) is provided. Therefore, the present invention is extremely useful in the medical field.

This application is based on Japanese Patent Application No. 2018-028329 (filing date: February 20, 2018) filed in Japan, the contents of which are all incorporated herein.

Claims (4)

A method for testing multiple sclerosis, comprising the step of measuring secretory receptor protein tyrosine phosphatase Z (PTPRZ) in a sample from a subject that is a brain section and/or cerebrospinal fluid , said subject If the measured amount of secretory PTPRZ in the sample from which the sample was derived is low compared to the amount of secretory PTPRZ in the control group sample, it is indicated that the subject has multiple sclerosis, and the secretory The method, wherein the PTPRZ is either or both secreted PTPRZ isoform 1 and secreted PTPRZ isoform 2. 2. The method according to claim 1, wherein said secretory PTPRZ isoform 1 and secretory PTPRZ isoform 2 are branched O-mannose glycosylated PTPRZ. Measurement of secretory PTPRZ in spinal fluid derived from a subject is performed using an antibody that specifically recognizes the protein or an antibody that specifically recognizes a branched O-mannose sugar chain, 3. A method according to claim 1 or 2 . A diagnostic kit for multiple sclerosis comprising an antibody that specifically recognizes secretory PTPRZ, wherein the secretory PTPRZ is either or both secretory PTPRZ isoform 1 and secretory PTPRZ isoform 2 A kit.

Images